Calcitriol, the hormonally active form of vitamin D3, has well known anti-cancer activity. We have recently identified four novel pathways of additional calcitriol actions that inhibit breast cancer (BCa) growth. (1) Calcitriol directly inhibits aromatase expression in BCa cells and adipose tissue thereby reducing local estrogen synthesis and the major growth stimulus to estrogen receptor (ER) positive BCa cells. Importantly, calcitriol exhibits tissue specific regulation of aromatase causing down-regulation in BCa and adipose tissue and up-regulation in bone. The latter action would protect bone from the side effect of osteoporosis due to estrogen deprivation routinely seen in aromatase inhibitor (AI)-treated patients. (2) Calcitriol inhibits the synthesis of prostaglandins (PG), major stimulators of BCa growth, by multiple actions including suppression of COX-2 expression. (3) PGs are potent inducers of aromatase transcription in BCa and calcitriol's inhibition of PGs also indirectly reduces aromatase expression. (4) Calcitriol directly down-regulates ER1 expression. Thus calcitriol acts by multiple pathways to reduce the levels of both the ligand (estrogens) and the receptor (ER) and thereby suppress BCa growth. We postulate that calcitriol and AI combination therapy will improve AI efficacy, potency and safety, reduce osteoporosis risk and delay the development of AI-resistance. Objective: We will study these four pathways of calcitriol action and evaluate the effectiveness of the combination of calcitriol with AIs compared to individual drugs. These studies will rapidly pave the way to clinical trials of calcitriol-AI combination therapy in BCa patients. Specific Aims: Aim I examines calcitriol regulation of aromatase and PG pathway genes in BCa, adipose, ovary and bone cells and the effect of the calcitriol-AI combination to inhibit BCa cell growth. Aim II studies the mechanism for the differential regulation of aromatase. Aim III determines the mechanism by which calcitriol down-regulates ER1. In vivo studies in Aim IV assess the effects of calcitriol, AIs and their combinations in xenograft mouse models on tumor growth and the development of AI-resistance. Regulation of aromatase expression will be examined in tissue-banked human BCa specimens from women treated with vitamin D3. Study Design: Calcitriol effects on aromatase and PG pathway genes and down-stream biological responses will be studied in BCa cell lines, primary normal breast and BCa cells and BCa xenografts in nude mice. Mechanistic studies will address differential regulation of aromatase promoters in BCa, adipose, ovary and bone cells and ER1 in BCa cells. The effects of calcitriol, AIs and combinations on BCa growth will be assessed in cell culture and animal models of BCa. Potential Benefits: The proposed studies will evaluate calcitriol, AIs and combinations for therapy of BCa patients. Calcitriol and AIs are approved drugs, orally active, safe and are of modest cost. We expect these findings to swiftly advance calcitriol-AI combinations to clinical trials to treat and/or prevent BCa. PUBLICHEALTH RELEVANCE We postulate that calcitriol plus an AI will show enhanced activity to prevent the growth and progression of BCa. We believe the data generated by this study will pave the way to support the rapid advancement of calcitriol-AI combination therapy to clinical trials for the treatment of BCa. This strategy will provide a safe and economical improvement to the usual therapeutic regimen administered to BCa patients.